Detection of neisseria meningitidis by loop mediated isothermal amplification

ABSTRACT

The invention provides a LAMP assay for detection of meningococcal disease, the test comprising at least one nucleic acid primer set capable of detecting  Neisseria meningitides  in a LAMP based molecular test, the primer set being chosen from the primer sets listed in Table 1 as LAMP SETS 1 to 12 comprising SEQUENCE IDs from ID: 1 to ID: 69. Each assay consists of a primer set including of one pair of forward (HP) and reverse (BIP) inner primers, forward (F3) and reverse (B3) outer primers. The assay may also include loop forward (LF) and/or loop back (LB) primers to accelerate the reaction.  Neisseria meningitides  serotypes A, B, C, Y and W135 can be detected using the assay of the invention.

The present invention relates to a test for meningitis. Morespecifically the invention relates to a near bedside assay for Neisseriameningitidis.

The pathogenic bacterium Neisseria meningitidis (NMG) is a majorworldwide cause of invasive bacterial meningitis and septicaemia, knownas ‘meningococcal disease’ (MD). Although relatively rare in developedcountries (1-3 cases per 100,000 population per year), MD has seriousconsequences for those who are affected, especially children.Vaccination has reduced the incidence of MD in recent years, although noeffective vaccine is available for Group B NMG, which causes >40% of UKcases. MD has a mortality rate of between 15% (meningococcal meningitis)and 50% (meningococcal septicaemia), despite the availability ofeffective antibiotic therapy. This is partly because of difficulty withdiagnosis of early-stage MD, which can lead to delayed diagnosis.

Diagnosis of MD currently relies entirely on correct interpretation ofclinical symptoms, which are frequently absent or equivocal. This makesclinical diagnosis challenging, especially in very young children, andthere is no reliable diagnostic test available to assist physicians withmaking a diagnosis of early-stage MD. Existing laboratory tests for NMGinfection involve either conventional isolation and culture—which takestoo long to be useful as part of a clinical diagnosis, or ‘polymerasechain reaction’ (PCR) tests—which can also be time-consuming, and areoften available only in reference laboratory settings. The most rapidcurrent test to detect NMG (gene-specific PCR) is not available in mosthospital bacteriology laboratories, due to lack of expertise/specialistequipment for molecular diagnostic testing. For this reason, thecurrently available tests are used only to confirm a clinical diagnosisof MD.

The consequences of delayed diagnosis for the patient can becatastrophic—leading to permanent disability (often in the form ofserious neurological damage or multiple limb amputations) or death.There is a clear need for a rapid and reliable molecular diagnostictest, offering high positive predictive value (PPV) and negativepredictive value (NPV) to assist with diagnosis of early-stage MD. Mostimportantly, a test which could be applied either at the point of care,or in a near-patient setting, and which gave a rapid result (1-2 hours)would provide physicians with timely information which is currently onlyuseful to confirm a diagnosis

Neisseria meningitidis is a major cause of bacterial meningitis andsepticaemia worldwide. Early diagnosis of meningococcal disease (MD) isdifficult because the initial presenting features are common to those ofsimple upper respiratory tract infection such as coryza and sore throat.Delayed diagnosis of MD can have catastrophic consequences for thepatient, and contributes to the high levels of morbidity and mortalitywhich can be associated with MD, especially in children. The classicalfeatures of MD, which include haemorrhagic rash, meningism and reducedlevel of consciousness, may come on rapidly but still relatively late(e.g. 12-24 hours) after the first symptoms of the illness start. Arecent study (reference 1) has suggested that leg pains, coldextremities and abnormal skin colour are seen in the first 12 hours ofMD. However, the positive and negative predictive value of these signsis not known, and these symptoms are likely to be present in childrenwith other infections such as influenza. Similar difficulties alsosurround early clinical diagnosis of meningitis and septicaemia causedby other bacteria, especially Streptococcus pneumoniae. Significantly,there is no reliable test to assist clinicians with diagnosis ofearly-stage MD; diagnosis can be made only on the basis of clinicalsymptoms, with inevitably serious consequences if these symptoms areabsent or overlooked.

Laboratory-based molecular tests are currently not useful as diagnostictools (except for confirming a diagnosis) because laboratory turn aroundtimes can be significant, even for the fastest tests. The transport timebetween specimen collection and laboratory testing also has asignificant adverse effect on positivity rates for confirmatory MDmolecular assays (reference 2), leading to false-negative test results.While laboratory culture of N. meningitidis has historically been thegold standard for confirming diagnosis of MD, pre-admission antibiotictherapy has greatly reduced the value of routine culture, and the timerequired (24-48 hours) generally limits its role to confirmingantibiotic susceptibility in culture-positive specimens. Data from aprevious study in our laboratory (reference 3) illustrates the very lowsuccess rate of culture in confirming diagnosis of MD.

In view of these problems, a rapid and reliable molecular diagnostictest, offering high positive predictive value (PPV) and negativepredictive value (NPV), would be extremely useful. Most importantly, atest which could be applied either at the point of care, or in anear-patient setting, and which gave a rapid result (1-2 hours) couldavoid the critical delays which are associated with submitting aspecimen for laboratory testing. This would assist clinicians byproviding information which is currently only available to confirm adiagnosis of MD.

A number of molecular tests for laboratory detection of N. meningitidishave been described in the literature. These tests generally rely on thePolymerase Chain Reaction (PCR) to amplify and detect virulence genes,and focus on identification of cultured N. meningitidis isolates(references 4 & 5) or detection using invasive specimens such as bloodor cerebrospinal fluid (references 6 & 7). In the course of a recentresearch project investigators demonstrated an effective combined PCRlaboratory assay which detects two important virulence genes from N.meningitidis in nose and throat swab specimens (Dr. K. Dunlop, MDThesis; reference 3). This assay proved to be very effective forconfirming diagnosis of MD in a case-control clinical trial (n=104suspected cases, n=104 case controls), which showed that the test hashigh sensitivity (81%), specificity (100%), PPV (100%) and NPV (92%).The study clearly showed that these gene targets are useful diagnosticbiomarkers of MD, and they can be easily detected in non-invasiveclinical specimens. Current recommendations from the Chief MedicalOfficer include taking blood samples for PCR analysis and nose/throatswabs for culture only. Published PHLS guidelines (reference 8) notethat molecular testing of throat swab specimens is effective, althoughthey do not consider this to be ‘definitively diagnostic’ forconfirmatory testing by reference laboratories at present. Nevertheless,recent data strongly suggest that the use of combined molecular testingdirectly on nasal and pharyngeal swab specimens has considerablepotential for improving the diagnosis of MD in this hospital andelsewhere.

While PCR assays are clearly a valuable laboratory diagnostic method, itwould not be practical to apply these tests in a near-patient setting,as they require both laboratory skills and specialised instruments (athermal cycler and gel electrophoresis equipment, or a ‘real-time’ PCRinstrument).

It is an aim of the present invention to develop a test which detectsthe same gene targets as the current PCR assay, but using an alternative‘isothermal’ DNA amplification technique, in a format which would bepractical for near-patient testing by staff without specialisedlaboratory skills.

It is an aim of the present invention to provide a rapid qualitativemolecular test to detect Neisseria meningitidis DNA in patientspecimens, allowing rapid confirmation of meningococcal infection.

There is currently no rapid diagnostic test which can be used to assistwith clinical diagnosis of early-stage meningococcal disease, andsignificant turnaround times are required even for the fastestlaboratory tests. In contrast, the proposed new test would be availablefor near-patient testing by staff without laboratory skills and withouthighly specialized or costly equipment (i.e. it could be used inhospital A&E/E.R. departments, large primary care units or pharmacies).

According to the present invention there is provided a diagnostic testfor meningococcal disease, the test comprising nucleic acid primer setscapable of detecting Neisseria meningitides in a LAMP based moleculartest, the primer set being chosen from the primer sets listed in Table 1as LAMP SETS 1 to 12 comprising a set of SEQUENCE IDs selected from ID:1 to ID: 69.

A LAMP primer set consists of one pair of forward (FIP) and reverse(BIP) inner primers, forward (F3) and reverse (B3) outer primers. FIP,BIP, F3 & B3 primers are essential for amplification to proceed. Theaddition of loop forward (LF) and/or loop back (LB) primerssignificantly accelerates amplification reducing overall detection timesby 50%

The invention also provides any of the primers as set out in Table 1 asSequence ID: 1 through Sequence ID: 69, individually or in combinationwith any of the other listed primers for use in an assay for Neisseriameningitides.

Preferably the assay is a LAMP assay.

Preferred primer sets are chosen from the group consisting of LAMP SETS1, 3, 5, 6, 7 and 12.

Particularly preferred primers sets are 3, 5, 1, 7, 12 and 6.

In a preferred embodiment of the invention the primer set is chosen fromLAMP primer sets 3 or 5 as shown in Table 1.

The nomenclature used herein to describe the primer sequences forpreferred LAMP sets is as follows. L3L3 refers to LAMP set 3 (FIP, BIP,F3 & B3) and loop set 3 i.e. —Seq IDs 11 to 14 and 16 and 17. L3L1refers to LAMP set 3 (FIP, BIP, F3 & B3) and loop set 1 i.e. —Seq IDs 11to 14 and 15 and 16. In each case the first 4 sequences are essentialand the additional two loop primers accelerate the reaction.

Preferably the LAMP primer set comprises SET 3 (L3L3) consisting of SeqIDs 11 to 14 and IDs 16 and 17.

An alternative preferred primer set comprises SET 3 (L3L1) consisting ofSeq IDs 11 to 16.

Another preferred primer set comprises SETS (L5L1) consisting of Seq IDs24 to 29.

The preferred primer sets are listed in Table 2

The invention therefore provides the use of the listed primers in adiagnostic test for Neisseria meningitides.

The invention will be further described with reference to the followingexperimental details and with reference to the accompanying FIGUREwherein

FIG. 1 illustrates Real Time ctrA LAMP plot total fluorescence againsttime in mins. for Neisseria meningitidis serogroups A NCTC10025,serogroup B NCTC10026, serogroup Y NCTC10791, Neisseria flavescens NCTC3191, Neisseria polysaccharea NCTC1858, Neisseria lactamica NCTC 10616,Neisseria cinerea NCTC 10294 total nucleic acid extracts & No TemplateControl (Nuclease Free Water)

The exemplification of the utility of the invention involves consecutivephases:

-   -   Phase 1—Assay development and laboratory optimisation    -   Phase 2—Transfer of the assay to a ‘near patient’ clinical        setting    -   Phase 3—Clinical validation of the near-patient assay    -   Phase 4—Data analysis and reporting

Phase 1—Assay Development and Laboratory Optimisation Target Selectionand Assay Design:

The proposed test detects the same gene targets as the existing PCRassay. These are: ctrA, encoding a capsule polysaccharide export outermembrane protein; and porA, encoding a separate outer membrane protein.The products of these genes are important virulence determinants in N.meningitidis, and the inventor's data demonstrates that the conservedregions within these genes are useful biomarkers of MD.

It would not be practical to apply existing PCR tests in a near-patientsetting so the inventors have developed a test which detects the sametargets, but uses an alternative ‘isothermal’ DNA amplificationtechnique. Unlike PCR, isothermal methods do not require expensive orcomplicated thermal cycling instruments, which makes them veryattractive for point-of-care or near-patient testing. An isothermaltechnique called ‘Loop-mediated isothermal Amplification’ (LAMP), usedboth in laboratory and near-patient settings has distinct advantagesover the current PCR laboratory test.

The LAMP method (references 9, 10 & 11) is a type of ‘stranddisplacement’ amplification, which utilises a specially designed set ofoligonucleotide primers, and a specific thermophilic DNA polymerasederived from Bacillus stearothermophilus. The primers are designed topromote the formation of ‘hairpin-loop’ structures during the initialstages of the reaction, allowing high levels of self-primed DNAsynthesis to occur from these structures as the reaction continues. Inbrief, the reaction is initiated by annealing and extension of a pair of‘loop-forming’ primers, followed by annealing and extension of a pair offlanking primers. Extension of these primers results instrand-displacement of the loop-forming elements, which fold up to formterminal hairpin-loop structures. Once these key structures haveappeared, the amplification process becomes self-sustaining, andproceeds at 60-65 degrees C. in a continuous and exponential manner(rather than a cyclic manner, like PCR) until all of the nucleotides(dATP, dTTP, dCTP & dGTP) in the reaction mixture have been incorporatedinto the amplified DNA. LAMP allows amplification of target DNAsequences with higher sensitivity and specificity than PCR, often withreaction times of below 30 minutes, which is equivalent to the fastestreal-time PCR tests. The target sequence which is amplified is typically200-300 base-pairs (bp) in length, and the reaction relies uponrecognition of between 120 bp and 160 bp of this sequence by severalprimers simultaneously during the amplification process. This high levelof stringency makes the amplification highly specific, such that theappearance of amplified DNA in a reaction occurs only if the entiretarget sequence was initially present. While characterisation of theamplified DNA (on the basis of its restriction pattern or DNA sequence)is possible, this is generally not necessary as the reaction is sospecific; the presence of amplified DNA indicates that the targetsequence was present. Significantly, the yield of amplified target DNAin positive reactions is so high, it can be easily and directly detectedin the reaction tube (references 12 & 13) allowing rapid discriminationbetween positive and negative specimens.

A number of diagnostic LAMP assays have been described in theliterature, including tests to detect causes of viral meningitis such asmumps virus (using RT-LAMP; reference 14) and human herpes virus 7(HHV-7; reference 15). The published HHV-7 LAMP assay was clinicallyvalidated, and used to detect primary HHV-7 infection in serum samplesin a 60 minute assay. Although there is growing interest in the use ofLAMP to develop rapid diagnostic tests, no studies using LAMP to detectN. meningitidis have been published to date.

The very high sensitivity of LAMP may allow the initial DNA extractionstep during the assay to be avoided, allowing a diluted specimen lysateto be used instead of purified DNA. This has the potential to furtherreduce the total time required to complete the test, as the extractionstage forms an increasingly large proportion of the total assay time asfaster amplification and detection methods are employed. A publishedallele-specific LAMP assay for human cytochrome P450 ‘single nucleotidepolymorphism’ genotyping (reference 17) has been used to selectivelyamplify target sequences from whole-blood lysates, which suggests thatthis approach is viable. The P450 study also demonstrated that LAMP canbe used to discriminate between closely related genotypes, which isrelevant for the N. meningitidis genotyping test (using the siaD gene)proposed here.

The inventors have now developed a prototype LAMP assay which can detectthe N. meningitidis etrA gene target in extracted clinical specimens in60 minutes using LAMP primers as set out in Table 1.

This initial part of the project involved detailed gene sequenceanalysis, followed by the design and testing of a range of LAMP primersets. The laboratory performance of each set has been examined, and themost effective and practical primers selected for transfer to a clinicalsetting (Phase 2) and clinical validation (Phase 3).

The designed primer sets are set out in Table 1 and identified as LAMPSETS 1 to 12 consisting of primers being sequence ID: 1 through tosequence ID: 69, together with an indication of their effectiveness withdifferent strains.

Assay Optimisation and Laboratory Validation:

The principal objective of this part of the study is an optimised androbust test which will be useable by a member of staff with minimaltechnical training. This work will compare LAMP with the existing nestedPCR test method, and will focus on the ctrA and porA targets, with thesiaD and lytA targets as secondary objectives. Laboratory validationwork will include:

-   -   Optimisation of the LAMP reaction conditions (by varying the        reagent composition, incubation temperature, reaction time        etc.).    -   Preparation of control and reference material (i.e. cloned        reference targets).    -   Determination of the analytical sensitivity of the test (using        reference materials).    -   Determination of clinical sensitivity (using ‘spiked’ and real        clinical samples).    -   Specificity testing, using typed N. meningitidis strains, other        clinically relevant bacteria, and human genomic DNA. The        specificity panel will include NCMBI reference strains from 9        serogroups known to cause invasive disease (A, B, C1+, C1−, X,        Y, W-135, Z & L) and clinical isolates from this hospital.        Identification of clinical isolates will be confirmed by 16S        sequencing.    -   Comparison of test performance using fresh vs. stored specimens.

A major objective is to understand possible sources of false-positiveand false-negative results, in order to maximize the PPV and NPV of thetest. A positive control LAMP assay will also be developed, allowingcontrol reactions to be included with every batch of unknowns. PhageLambda DNA will be used as a control target, as this is readilyavailable, easily standardized, and will not interfere or cross-reactwith detection of any of the intended target genes.

As an additional benefit of this project, it should be noted that theoptimised and laboratory-validated tests will be immediately useful forrapid identification of N. meningitidis and S. pneumoniae isolates aspart of the routine clinical microbiology service in this hospital, andelsewhere.

Real-Time Detection Methods:

Real-time detection of amplified DNA during LAMP reactions is possible,using three different methods: by turbidometry (detection of insolublemagnesium pyrophosphate accumulation in reactions), by ethidium bromidefluorescence, or by SYBR Green I fluorescence (which both detectaccumulation of double-stranded DNA in reactions). Real-time monitoringincreases the complexity of the test/instrument format required, as someform of optical or fluorescence measurement must be used. However, thisis offset by the fact that provisional positive results may be obtainedmuch more quickly (i.e. as soon as an amplified product is detected in areaction). Detectable amounts of DNA may be synthesised in a little as15 minutes in LAMP reactions (reference 18) and in some cases thepositive reaction can even be visualized by eye.

Following the assay optimisation stage, the proposed project will alsoassess the available real-time monitoring methods, to see whether theycould usefully be incorporated into a near-patient test.

Extraction Methods:

An important part of the study will be the development of a rapid DNAextraction method which can be used for near-patient testing. Currentlaboratory protocols for DNA extraction are too cumbersome for use innear-patient settings, so a simpler method involving specimen lysis anddilution will also be developed, using both PCR and the optimised LAMPtest to assess performance. The inventors are investigating whether theuse of crude specimen lysates for molecular assays is feasible, in orderto simplify the process further, and to minimise the specimen processingtime required.

Sequencing:

Some DNA sequencing work is being conducted for two reasons:

-   -   i) to confirm the taxonomic position of N. meningitidis isolates        obtained during the study, and previously isolated reference        strains (by 16S rDNA sequencing). This will rule out        misidentification of the putative pathogen in cases where        near-patient and/or laboratory molecular tests to detect N.        meningitidis prove to be negative.    -   ii) to increase the number of ctrA, porA and siaD sequences in        the database. This will allow more detailed sequence analysis to        be undertaken, and the design of primer sets to be refined        further, especially for genotyping purposes.

Only the optimised N. meningitidis ctrA and porA LAMP assays will beused for subsequent near-patient testing.

Phase 2—Transfer of (ctrA and porA) Tests to Near-Patient Setting

Protocol Development:

Some additional method development is expected, both to deliver a usablenear-patient testing protocol, and for the comparative laboratorytesting protocols. In particular, the issues surrounding specimencollection and processing will be addressed at this stage, and detailed‘standard operating procedures’ will be written. All specimens will besubjected to culture through the routine bacteriology service, andspecimens will be processed in parallel using the previously developedPCR assay and the optimised near-patient method in a laboratory setting,for comparison.

Training:

As a central objective of the proposed work is to develop a test whichcan be used by non-laboratory staff, appropriate training, supervisionand mentoring will be provided for the non-technical staff (researchnurses) who will conduct the test in a near-patient setting. The highsensitivity of molecular tests makes them susceptible to contaminationby amplified test products, which can lead to false-positive results. Animportant part of the training will be to ensure that the staff involvedunderstand the issues surrounding contamination, and can avoidcontamination of the near-patient testing area. The final assay willincorporate negative control reactions at all stages so thatcontamination problems can be quickly identified and addressed.

Pre-Clinical Validation:

Pre-clinical validation of the optimised test will be essential toassess the performance of assay in a near-patient setting. This phasewill therefore conclude with blinded and randomized processing of anumber of spiked and control specimens, to confirm that the newprotocols can be used as anticipated.

Phase 3—Clinical validation

Study Design: Modified Case-Control. Patient Groups

Group 1: All children with suspected meningococcal disease (MD) areentered into a ‘clinical care pathway’ and have a standardised set ofinvestigations (to make diagnosis, assessment of severity and initialtreatment) performed. In the recently completed one year study in theinventors' unit, 104 suspected MD children were recruited in 12 monthsand over 33% had proven MD (clinical picture PLUS blood culture +ve or+ve meningococcal PCR at Manchester Reference Laboratory). The inventorswill perform a case control study over at least a 2½ year period. Thenew test (measured from a combined nasal and throat swab, and blood)would be applied to all children with suspected MD entering the MD carepathway (estimated N=250 children) over this period (giving about 80definite cases). This group of children is already ‘filtered as possiblecases of MD’. The inventors want to be sure that the newly developedtest has a high sensitivity. If the true sensitivity were 90% then astudy of 80 affected children would give an estimate of the sensitivitywith 95% exact confidence intervals of width no wider than 81% to 95%.Children entered into this group would include those in whom the A&Edoctor considered might possibly have MD (fever, petechial rash or signsof meningism and those with signs of possible septicaemia—eg. featuresof circulatory failure). These children routinely have a ‘meningococcalpack’ performed (blood cultures, serology, PCR for reference laboratory,blood count and ESR, CRP) and a nasal and pharyngeal swab taken for PCRand culture. In this study an additional combined nasal and throat swabwill be taken from every child entered into this MD care pathway.

Group 2: The inventors want their newly developed test (assessed on acombined nasal and throat swab, and blood where possible) to have a veryhigh specificity/NPV. If the true specificity was 98% then a study of750 unaffected children would give an estimate of the specificity with95% exact confidence intervals of width no wider than 97% to 99%. Theyplan to study 750 children attending the A&E department withnon-specific febrile or upper respiratory tract illnesses who are notbeing entered into the meningococcal care pathway.

Included in this group will be children with;

i] simple febrile illnesses with features of a head cold (excludingchildren with classical respiratory infections eg ‘croup’)ii] non-specific fevers including those with leg pains, cold hands andfeet but who are not considered to be ‘ill’ to enter into the MD carepathway which could include those with early features of sepsis. Suchpatients were described recently as risk factors for early MD(Reference 1) but the frequency of these symptoms in the non-meningitispopulation is not known. A combined throat and nasal swab for the new MDtest will be taken from these children by the researchers, along with ablood sample, where this is possible. As most of these children willlikely be sent home from A&E a follow-up telephone call will be made 24,48 and 72 hours after recruitment to determine the natural resolution(or not) of the illness.

In conclusion design and testing of new LAMP primer sets has beendeveloped and the results are shown in detail in Table 1. LAMP reactionswill be optimized together with development of rapid extractionprotocols for clinical specimens (nasal and pharyngeal swabs).

Comparison of LAMP and PCR assays with respect to specificity,analytical/clinical sensitivity, time-to-result, and practicality fornear-patient use, using both standard and rapid extraction protocolswill be undertaken to demonstrate rapid and robust laboratory validatedassays which can detect the target genes with high specificity and knownanalytical sensitivity.

This invention will provide rapid laboratory assays for N. meningitidisdetection and genotyping, and rapid diagnostic tests for detection of N.meningitidis in non-invasive specimens, validated in both laboratory andclinical settings, which will be effective enough to be widely adoptedin near-patient settings.

A LAMP assay to detect the lytA gene (encoding the protein autolysin)from Streptococcus pneumoniae, has been described previously (reference16). This laboratory assay was shown to detect S. pneumoniae DNA withhigh specificity, and a sensitivity 10³ times higher than conventionalsingle-round PCR. As a secondary objective, the inventors propose tovalidate this published assay in the laboratory alongside the new testduring this study, for testing of ctrA and porA negative specimens. Thiswill also enable them to collect preliminary data on whether moleculardetection of S. pneumoniae in nasal or throat swab specimens mightcorrelate with diagnosis of pneumococcal meningitis or septicaemia inthe study group.

Results LAMP Primer Design

Oligonucleotide primers specific for N. meningitidis ctrA gene weredesigned corresponding to recognised conserved genomic regions(Positions in ctrA gene AF520902.1) using online LAMP primer designsoftware Primer Explorer version 3.0 available athttp://loopamp.eiken.co.jp/e/lamp/primer.html (NB. LAMP & LOOP primerset ID: 1 was designed by one of the inventors without aid of software).A LAMP primer set consisted of one pair of forward (FIP) and reverse(BIP) inner primers, forward (F3) and reverse (B3) outer primers. FIP,BIP, F3 & B3 primers are essential for amplification to proceed. Theaddition of loop forward (LF) and/or loop back (LB) primerssignificantly accelerates amplification reducing overall detection timesby 50% (Nagamine, et al, 2002) and this was confirmed by data generatedin the inventors' laboratory. With certain LAMP primer sets it waspossible to design only Loop back (LB) primers (eg. LAMP6, LAMP4) andfor others no forward or back Loop primers could be designed. See Table1 for list of primer sets.

The primers are designed to promote the formation of ‘hairpin-loop’structures during the initial stages of the reaction, allowing highlevels of self-primed DNA synthesis to occur from these structures asthe self sustaining reaction proceeds (Notomi, et al, 2000). Aby-product of LAMP reactions is magnesium pyrophosphate which can bemeasured by turbidity/fluorescence endpoint or in Real Time (Tomita, etal, 2008). Animation of the reaction (minus loop primers) can be seen athttp://loopamp.eiken.co.jp/e/lamp/anim.html and the loop principle isoutlined by Nagamine, et al, 2002 and athttp://loopamp.eiken.co.jp/e/lamp/loop.html.

Neisseria Meningococcus ctrA LAMP Primer Sets

All primer sets designed using LAMP software July 2007, except ID: 1designed March 2006). Core sequences shown in bold.

Primers: FIP=forward inner; BIP=backward inner; F3=forward outer;B3=backward outer; LF=loop forward; LB=loop backward.Positions in ctrA gene AF520902.1

TABLE 1  ctrA LAMP primer sets and Sequence ID numbers. ctrA PRIMERSEQUENCE 5′-3′ LAMP SET SEQ ID SET 1 ID1 FIPCGTCTATGGGTGCGGTGGGGAGACGATCTTGCAAACCGCCCATAC ID2 BIPGTAACCACATCACCGCGACGCAGCATGTGCAGCTGACACGTGGCAATG ID3 F3CCACGCGCATCAGAACGG ID4 B3 CGGCAAATGTGCAGGATACGA ID5 LF1GCTTATCGCTTTCTGAAGC ID6 LB1 GCAACTAAATCTTCCAAGGC SET 2 ID7 FIPATCACCGCGACGCAGCAAAATAAGTACGAACTGTTGCCTTGG ID8 BIPACCTTTACGTCTATGGGTGCGGAAGCCTCTYGCTGAAAAACC ID9 F3 GCTGACACGTGGCAATGTID10 B3 CCAATGGCTTCAGAAAGCGA SET 3 ID11 FIPCAAACACACCACGCGCATCAGATCTGAAGCCATTGGCCGTA ID12 BIPTGTTCCGCTATACGCCATTGGTACTGCCATAACCTTGAGCAA ID13 F3 AGCYAGAGGCTTATCGCTTID14 B3 ATACCGTTGGAATCTCTGCC ID15 LF1 CGATCTTGCAAACCGCCCA ID16 LB1 & LB3GCAGAACGTCAGGATAAATGGA ID17 LF3 CGATCTTGCAAACCGCCC SET 4 ID18 FIPCAAACCGCCCATACGGCCAAATCGGTTTTTCAGCYAGAGG ID19 BIPAAGATCGCCGTTCTGATGCGCCGTTCTGCCGGCAATTCC ID20 F3 CGGTGGGGAGAACACAAGID21 B3 ACTGCCATAACCTTGAGCAA ID22 LB1 GTGGTGTGTTTGTGTTCCGCTAT ID23 LB2GTGGTGTGTTTGTGTTCCGCTATA SET 5 ID24 FIPCACCACGCGCATCAGAACGGCAGCYAGAGGCTTATCGC ID25 BIPTGTTCCGCTATACGCCATTGGTTGCCTCACTGCCATAACCT ID26 F3 CGGTGGGGAGAACACAAGID27 B3 GCGCATCAGCCATATTCACA ID28 LF1 & LF3 CGGCCAATGGCTTCAGAAA ID29 LB1GGAATTGCCGGCAGAACGTC ID30 LB3 GAATTGCCGGCAGAACGTC SET 6 ID31 FIPTCCCCACCGCACCCATAGACCGGTGATGTGGTTACCATGA ID32 BIPATCGGTTTTTCAGCYAGAGGCTTTGCAAACCGCCCATACG ID33 F3 AGTTGCAAATCCGCGACAAID34 B3 CGCATCAGAACGGCGATC ID35 LB1 ATCGCTTTCTGAAGCCATTGG ID36 LB2TCGCTTTCTGAAGCCATTGG SET 7 ID37 FIPGCGAATGCGCATCAGCCATATTTGCTCAAGGTTATGGCAGTG ID38 BIPTTGTATGTGTCGAATGCGCCGTCGGCGAGAACACAAACGA ID39 F3 GAATTGCCGGCAGAACGTID40 B3 ATACTGTTCGCGCCACTG ID41 LF1 & LF2 CACGATATACCGTTGGAATCTCTGID42 LB1 TGGCTGAAGTGCAGAAATTCTT ID43 LF6 ACACGATATACCGTTGGAATCTCTID44 LB2 & LB6 TGGCTGAAGTGCAGAAATTCTTG SET 8 ID45 FIPCCATCACTTGTGGCTGATTGGCGGTCGGTAAAACGCCTGG ID46 BIPGGCGAATGTGTCGGTGATTCGTGCATCCAACACACGCTCA ID47 F3 TGCCGTTTGTTGGCGATAID48 B3 CACATTTGCCGTTGAACCAC SET 9 ID49 FIPGGCGTTTTACCGACCACCGAGGCACGTGGTACGGTTTC ID50 BIPAGGCCGCCTGAAAAAAATGGCCGACACATTCGCCGCATTA ID51 F3 AGTTGCCAGAGCAGTTGGID52 B3 CGCACACTATTCCCAGCAC SET 10 ID53 FIPCACCACGCGCATCAGAACGGCAGCYAGAGGCTTATCGC ID54 BIPTGTTCCGCTATACGCCATTGGTTGCCTCACTGCCATAACCT ID55 F3 CGGTGGGGAGAACACAAGID56 B3 GCGCATCAGCCATATTCACA ID57 LF1 CGGCCAATGGCTTCAGAAA ID58 LB1GGAATTGCCGGCAGAACGTC SET 11 ID59 FIPGGCCATTTTTTTCAGGCGGCCTTGGCGATATTTCGGTGGTC ID60 BIPCAAGTGATGGTGCGTTTGGTGCAGCGGCATACGCACACTA ID61 F3 ACGTGGTACGGTTTCTGTGID62 B3 CCACCGCATCCAACACAC SET 12 ID63 FIPCAACACACGCTCACCGGCTGGGCGAATGTGTCGGTGATT ID64 BIPGCGGTAGGTGGTTCAACGGCACTACATTGCCACGTGTCAG ID65 F3 GGTGCGTTTGGTGCAGAAID66 B3 TTCCAAGGCAACAGTTCGT ID67 LF1 CGTGCTGGGAATAGTGTGCGTID68 LB1 & LB2 ATGTGCAGGATACGAATGTGC ID69 LF2 GGSAATAGTGTGCGTATGCCGDegenerate bases key(Y = C/T) (S = G/C)ctrA LAMP Optimisation

The optimal operating temperature for each designed ctrA LAMP & LOOP set(60 to 65° C. inclusive) was determined by testing against N.meningitidis serogroup A strain NCTC10025 (NmA NCTC 10025) & N.meningitidis serogroup B clinical isolate 57/07 (NmB 57/07) QIAGEN totalDNA extract ten fold dilutions (NB. LAMP sets 2, 8, 9 & 11 excluded dueto inability to design corresponding Loop primers additionally LAMP 4was not assessed). See Table 2 for ranking.

LAMP&LOOP primer sets were ranked based upon the following criteria;

i] Visual Sensitivity—presence of visible colour change and turbidityindicating positive reaction ie. The lowest Cut off point for NmB 57/07& NmA NCTC 10025 detectable by eye.ii]. Speed of LAMP detection by Real Time analysis carried out onApplied Biosystems Real Time PCR instrument ABI7000 (ie Quickest ctrALAMP for initial detection and time to reach fluorescentplateau/maximum—occurs—10 mins after first fluorescent signal isgenerated see FIG. 1)iii]. ABI7000 Sensitivity (ie lowest cut off point as determined by RealTime LAMP)

TABLE 2 N. meningitidis ctrA LAMP & LOOP primer set ranking (bestperforming No. 1 etc) with optimal operating temperature, cut off point(ctrA copies detected per reaction) & time taken in minutes to reachmaximum fluorescence/Turbidity for NmA NCTC10025 & NmB 57/07 dilutions.Final Reaction volume = 25 μl (Specimen addition = 2.5 μl/LAMP MastermixVol. = 22.5 μl) ctrA COPY NO. CUT OFF PER TIME (MINS) TO REACH LAMP & 25μl REACTION MAX. FLUORO. RANK LOOP SET OPTIMAL TEMP. NmA NmB NmA NmB 1L3L1 63° C. 96.6 118 50 mins 40 mins 2 L3L3 61° C. 96.6 118 50 mins 49mins 3 L5L1 60° C.-61° C. 96.6 118 53 mins 60 mins 4 L13L1 61° C. 1.1 ×10³ 1.1 × 10³ 47 mins 41 mins 5 L5L3 62° C. 96.6 1.1 × 10³ 40 mins 49mins 6 L12L2 64° C. 1.3 × 10³ 1.1 × 10³ 60 mins 60 mins 7 L1L1 63° C.1.6 × 10⁴ 1.1 × 10³ 55 mins 57 mins 8 L12L1 62° C. 1.6 × 10⁴ 1.6 × 10⁴51 mins 51 mins 9 L7L6 60° C. 1.6 × 10⁴ 1.6 × 10⁴ 52 mins 50 mins 10L7L1 61° C. 1.6 × 10⁴ 1.6 × 10⁴ 60 mins 57 mins 11 L7L2 60° C. 1.3 × 10³1.6 × 10⁴ 60 mins 55 mins 12 L6 (no loop) 65° C. 2.1 × 10⁵ 1.7 × 10⁵ 60mins 60 mins

Based upon sensitivity ctrA LAMP3LOOP1 (L3L1), LAMP3LOOP3 (L3L1) &LAMP5LOOP1 (L5L1) had same cut off points. Repeat investigations withL3L1, L3L3 & L5L1 confirmed findings above.

The three best performing ctrA sets L3L1, L3L3 & L5L1 were chosen forfurther evaluation using N. meningitidis 57/07 spiked blood specimens.Investigations were carried out to determine if increasing overallspecimen addition volume from 2.5 μl to 5 μl, 10 μl and 12 μl andincorporation of a prior preheat denaturation stage of 95° C. for 5 minsfor specimens followed by immediate cooling on ice would improve overallsensitivity (protocol for prior heat denaturation published by Kamachi,et al, 2006 with impressive results). Results indicated that L3L1preheat 95° C./5 mins in combination with a 5 μl specimen additionprovided greatest sensitivity and reaction speed capable of detecting 28ctrA gene copies per reaction in less than 40 minutes. See Table 3. L3L1with prior specimen heat denaturation plus 5 μl specimen addition waschosen for clinical validation.

TABLE 3 Effect of varying specimen addition volume and heat denaturation95° C./5 mins of NmB 57/07 spiked blood specimens on analyticalsensitivity of L3L1, L3L3 & L5L1 and time in minutes required to reachmaximum fluorescence. Specimen Addition Volume 2.5 μl 5 μl 10 μl 12 μlPreheat Preheat Preheat Preheat 95° C./5 mins No Heat 95° C./5 mins NoHeat 95° C./5 mins No Heat 95° C./5 mins No Heat L3L1 175 1.7 × 10³ 28350 700 6.8 × 10³ 8.2 × 10³ 8.2 × 10³ ctrA copies ctrA copies ctrAcopies ctrA copies ctrA copies ctrA copies ctrA copies ctrA copies (32mins) (38 mins) (38 mins) (47 mins) (50 mins) (44 mins) (30 mins) (41mins) L3L3 175 175 28 3.4 × 10³  56 700 67 67 ctrA copies ctrA copiesctrA copies ctrA copies ctrA copies ctrA copies ctrA copies ctrA copies(35 mins) (36 mins) (45 mins) (52 mins) (34 mins) (52 mins) (34 mins)(49 mins) L5L1 175 1.7 × 10³ 350  3.4 × 10³ 700 5.2 × 10⁴ 8.2 × 10³ 8.2× 10³ ctrA copies ctrA copies ctrA copies ctrA copies ctrA copies ctrAcopies ctrA copies ctrA copies (35 mins) (45 mins) (38 mins) (47 mins)(51 mins) (34 mins) (35 mins) (41 mins)

Specificity

Total nucleic acid extractions from a total of 70 bacterial clinical andreference strains from 39 different bacterial species and 1 fungalreference strain were used in the present study to determine LAMP assayspecificity. All designed ctrA LAMP & LOOP primers specificallyamplified DNA from all N. meningitidis NCTC type strains and from 10clinical isolates of N. meningitidis serogroup B. There was no crossreactivity with other Neisseria species (n=6) tested or with 54different bacterial & fungal targets outlined in Table 4. ctrA LAMP &LOOP primers were 100% specific for capsular N. meningitidis strainstested. See FIG. 1 for example of specificity.

LAMP SETS 1, 2, 4, 7, 10 exhibited specificity for all NMG serotypestested (A, B, C, Y, W135). SETS 8, 9, 11 will not detect NmA beingspecific for B, C, Y, W135 only. SETs 3, 5, 6, 10 detect all serotypestested with wobble. SET 3 is non-A specific with C in wobble position orA specific with T in wobble position. Set 5 is non-A specific with C inwobble position, A specific and 29E with T in wobble position. Set 12exhibits specificity for types B, C, Y, W135 with either loop primer setbut detects A only using LB1/LF2 loop primers.

TABLE 4 List of bacterial and fungal species tested to determinespecificity of LAMP & LOOP primer sets. SPECIES STRAIN SEROGROUPNeisseria meningitidis NCTC 10025 A Neisseria meningitidis NCTC 10791 YNeisseria meningitidis NCTC 11203 W135 Neisseria meningitidis NCTC 8554C Neisseria meningitidis NCTC 10792 Z Neisseria meningitidis NCTC 10790X Neisseria meningitidis NCTC 11202 29E Neisseria meningitidis NCTC10026, B 531/07, 44/06, 217/06, 1386/06, 839/06, 368/05, 338/05, 57/07,1069/07, 304/06 Neisseria gonorrhoeae ATCC 49226 Neisseria sicca 920/06Neisseria. flavescens NCTC 3191 Neisseria lactamica NCTC 10616 Neisseriacinerea NCTC 10294 Neisseria polysaccharea NCTC 1858 Haemophilusinfluenzae NCTC 4560, 016/07 B Klebsiella pneumoniae NCTC 10896, 45/07Enterococcus faecalis 009/07 Enterobacter aerogenes NCTC 10006, 001/07Enterobacter cloacae NCTC 9394 Staphylococcus aureus ATCC 25923, ATCC29213, NCTC 10442, 09/07, M04/0071, HT2000/0132 Staphylococcuslugdunensis RVH Isolate Staphylococcus capitis RVH IsolateStaphylococcus sciuri ATCC 29061 Streptococcus pneumoniae NCTC 7465,NCTC 7978, RVHType 2, 59/07 Streptococcus parasanquis 74/07Streptococcus intermedius 305/07 Micrococcus luteus 290/07 Serratiamarcescens NCTC 10211, 287/07 Acinetobacter baumanii 36/07 Moraxellacatarrhalis RVH01, RVH09 Escheria coli NCTC 9001, ATCC 25922 Pseudomonasaeroginosa 46/07 Stenotrophomonas maltophilia ATCC 17666Peptostreptococcus spp. 6004-4 Bacteriodes fragilis CAH 6046-4Clostridium difficile CAH 72 Sphingobacterium spiritivorum ATCC 33861Sphingobacterium thalpophilum 1192/07 Acinetobacter zwoffii 98/07Klebsiella oxytoca ESBL+ 135/07 Burkholderia multivorans BCH IsolateProteus vulgaris 564/07 Bordetella parapertussis BCH Isolate Coxiella.burnetii 9 Mile Strain Citrobacter freundii NCTC 9750 Aeromonashydrophilia 659 Candida albicans ATCC 10028

Clinical Validation

A breakdown of L3L1 Clinical Validation results to date are outlined inTables 5 & 6 below. All specimens underwent total nucleic acidextraction using commercially available manual Qiagen QIAamp® kit.

TABLE 5 Using ctrA Real Time Taqman PCR Assay (Corless et al, 2001) asGold Standard for all Specimen Types Tested GOLD STANDARD POSITIVENEGATIVE TEST POSITIVE TP = 24 FP = 3 NEGATIVE FN = 0 TN = 233

Lamp Sensitivity Versus Taqman

Sensitivity=tp/(tp+fn)24/(24+3)×100=88.9%

In reality 2 LAMP False Positives are true positives. These were notidentified by Gold Standard Taqman PCR but had been found to be positiveby Manchester Reference Lab. PCR (MPCR). Considering thissensitivity=96%. One specimen was Taqman negative but repeatably LAMPpositive. It is possible that this individual was harboring a capsularmeningococcus (ie. Carrier state) or had low grade infection.

Lamp Specificity

Specificity=tn/(fp+tn)233/(3+233)×100=98.7%

(Again two False positives are in reality true positives taking thisinto consideration Specificity=100%)

TABLE 6 Specimen Type Breakdown % LAMP Positive NO. NO. LAMP SPECIMENTYPE TESTED POSITIVES (%) WHOLE BLOOD (B) 73 7 (9.6%) THROAT SWAB (TS)76 8 (10.5%) EDTA BLOOD (EDTA) 53 3 (5.7%) SWABS (SW) 13 2 (15.4%)CEREBROSPINAL FLUID (CSF) 9 0 (0%) SERA (S) 19 4 (21.1%) FAECES SOLID(FS) 2 1 (50%) FAECES (F) 1 0 (0%) FAECES SEMI SOLID (FSS) 1 0 (0%)NASAL SECRETIONS (NASE) 6 0 (0%) SECRETIONS (SEC) 3 1 (33.3%) TRACHEASECRETIONS (TRSE) 1 1 (100%) VIRAL SWAB (VIS) 1 0 (0%) SPUTUM 1 0 (0%)TOTALS 259 27 (10.4%)Mean time (mins) to reach maximum Fluorescent intensity=28.6 minsRange of detection=6.6×10⁶-4×10³ ctrA copies per ml (3.3×10⁴-20 ctrAcopies per reaction)

Improved Accuracy of Diagnosis by LAMP on Upper Airway Secretions

Using a Likelihood Ratio analysis, based on the prevalence of truemeningococcal disease in children admitted to the meningococcal carepathway in the Children's Hospital Belfast, combined with a LAMPsensitivity of 92% and specificity of 97% (allowing for a carriage rateof 2% in this age range) the Odds Ratio changes from 1:3 by clinicaldiagnosis to 10:1 by a LAMP diagnosis. This increased utility underliesthe value of undertaking a LAMP test on upper respiratory secretionsi.e. throat and nasal swabs.

Accordingly the present invention provides a series of near bedside realtime LAMP assays for Neisseria meningitidis with varying specificitiesbased on the LAMP sets and Sequence IDs set out in Table 1 and detailedin the following Sequence Listing prepared according to Patentin 3.5.

The assays of the invention can be used for early detection of Nmeningitides in samples which as easy to obtain and allow treatment ofthe individual to be tailored accordingly.

REFERENCES

-   1. Thompson M J, Ninis N, Perera R, Mayon-White R, Phillips C,    Bailey L, Hamden A, Mant D, Levin M. 2006. Clinical recognition of    meningococcal disease in children and adolescents. Lancet    367:397-403-   2. Sadler F, Borrow R, Dawson M M, Kaczmarski E B, Cartwright K, Fox    A J. 2000. Improved methods of detection of meningococcal DNA from    oropharyngeal swabs from cases and contacts of meningococcal    disease. Epidemiol Infect. 125:277-283-   3. Dunlop K, O'Neill H, McCaughey C, Curran T, De Ornellas D,    Mitchell F, Mitchell S, Feeney S, Wyatt D, Forde M, Shields M,    Jackson P, Coyle P. 2005. The role of common respiratory viral    infections in triggering invasive meningococcal disease. Eur Soc    Clin Virology 8^(th) Annual meeting, Geneva. Poster 38-   4. Lewis C, Clarke S C. 2003. Identification of Neisseria    meningitidis serogroups Y and W135 by siaD nucleotide sequence    analysis. J Clin Microbiol. 41:2697-2699-   5. Bennett D E, Cafferkey M T. 2006. Consecutive use of two    multiplex PCR-based assays for simultaneous identification and    determination of capsular status of nine common Neisseria    meningitidis serogroups associated with invasive disease. J Clin    Microbiol. 44:1127-1131-   6. Xu J, Moore J E, Millar B C, Webb H, Shields M D, Goldsmith    C E. 2005. Employment of broad range 16S rDNA PCR and sequencing in    the detection of aetiological agents of meningitis. New Microbiol.    28:135-143-   7. Molling P, Jacobsson S, Backman A, Olcen P. 2002. Direct and    rapid identification and genogrouping of meningococci and porA    amplification by LightCycler PCR. J Clin Microbiol. 40:4531-4535-   8. Public Health Laboratory Service Meningococcus Forum. 2002.    Guidelines for public health management of meningococcal disease in    the UK. Communicable Disease and Public Health Report 5:187-204-   9. Notomi, T, Okayama H, Masubuchi H, Yonekawa. T, Watanabe K,    Nobuyuki A, Hase T. 2000. Loop-mediated isothermal amplification of    DNA. Nucleic Acids Res. 28:e63-   10. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. 2001.    Loop-mediated isothermal amplification using a nondenatured    template. Clin Chem. 47:1742-1743-   11. Nagamine K, Hase T, Notomi T. 2002. Accelerated reaction by    loop-mediated isothermal amplification using loop primers. Mol Cell    Probes 16:223-229-   12. Soliman H & El-Metbouli, M. An inexpensive and rapid diagnostic    method for Koi Herpesvirus (KHV) infection by loop-mediated    isothermal amplification. 2005. Virology Journal 2:83-87-   13. Mori Y, Hirano T, Notomi T. 2006. Sequence specific visual    detection of LAMP reactions by addition of cationic polymers. BMC    Biotechnology 6:3-7-   14. Okafuji T, Yoshida N, Fujino M, Motegi Y, Ihara T, Ota Y, Notomi    T, Nakayama T. 2005. Rapid Diagnostic Method for Detection of Mumps    Virus Genome by Loop-Mediated Isothermal Amplification. J Clin    Microbiol. 43:1625-1631-   15. Yoshikawa T, Ihira M, Akimoto S, Usui C, Miyake F, Suga S,    Enomoto Y, Suzuki R, Nishiyama Y, Asano Y. 2004. Detection of Human    Herpesvirus 7 DNA by Loop-Mediated Isothermal Amplification. J Clin    Microbiol. 42: 1348-1352-   16. Seki M, Yamashita Y, Torigoe H, Tsuda H, Sato S, Maeno M. 2005.    Loop-Mediated Isothermal Amplification Method Targeting the lytA    Gene for Detection of Streptococcus pneumoniae. J Clin Microbiol.    43:1581-1586-   17. Iwasaki M, Yonekawa T, Otsuka K, Suzuki W, Nagamine K, Hase T,    Tatsumi K I, Horigome T, Notomi T, Kanda H. 2003. Validation of the    Loop-Mediated Isothermal Amplification Method for Single Nucleotide    Polymorphism Genotyping with Whole Blood. Genome Letters 2:119-126-   18. Parida M, Horioke K, Ishida H, Dash P K, Saxena. P, Jana A M,    Islam M A, Inoue S, Hosaka N, Morita K. 2005. Rapid Detection and    Differentiation of Dengue Virus Serotypes by a Real-Time Reverse    Transcription-Loop-Mediated Isothermal Amplification Assay. J Clin    Microbiol 43:2895-2903-   19. Bossuyt P M, Reitsma J B, Bruns D E, Gatsonis C A, Glasziou P P,    Irwig L M, Lijmer J G, Moher D, Rennie D, de Vet H C W (for the    STARD Group). 2003. Towards Complete and Accurate Reporting of    Studies of Diagnostic Accuracy: The STARD Initiative. Clin Cheri.    49:1-6-   20. Moher D, Schulz K F, Altman D G (for the CONSORT Group) 2001.    The CONSORT statement: revised recommendations for improving the    quality of reports of parallel-group randomised trials. Lancet    357:1191-1194-   21. Corless, C. E., Guiver, M., Borrow, R., Edwards-Jones, V.,    Fox, A. J. & Kaczmarski, E. B. (2001). Simultaneous Detection of    Neisseria meningitidis, Haemophilus influenzae and Streptococcus    pneumoniae in suspected Cases of Meningitis and Septicemia Using    Real Time PCR. Journal of Clinical Microbiology 39:1553-1558.-   22. Kamachi, K., Toyoizumi-Ajisaka, H., Toda, K., Soeung, S. C.,    Sarath, S., Nareth, Y., Horiuchi, Y., Kojima, K., Takahashi, M. &    Arakawa, Y. (2006). Development and Evaluation of a Loop Mediated    Isothermal Amplification Method for Rapid Diagnosis of Bordetella    pertussis Infection. Journal of Clinical Microbiology 44:1899-1902.-   23. Tomita, N., Mori, Y., Kanda, H. & Notomi, T. (2008).    Loop-mediated isothermal amplification (LAMP) of gene sequences and    simple visual detection of products. Nature Protocols 3:877-882.

1. A LAMP assay for detection of meningococcal disease, the testcomprising at least one nucleic acid primer set capable of detectingNeisseria meningitidis in a LAMP based molecular test, the primer setbeing chosen from the primer sets listed in Table 1 as LAMP SETS 1 to 12comprising SEQUENCE IDs from ID: 1 to ID:
 69. 2. An assay as claimed inclaim 1 wherein the primer set consists of one pair of forward (FIP) andreverse (BIP) inner primers, forward (F3) and reverse (B3) outerprimers.
 3. An assay as claimed in claim 1 further comprising loopforward (LF) and/or loop back (LB) primers.
 4. A LAMP assay as claimedin claim 1 for detection of Neisseria meningitidis, the assay usingprimer LAMP SET L3L3 consisting of Seq IDs 11 to 14, 16 and
 17. 5. ALAMP assay as claimed in claim 1 for detection of Neisseriameningitidis, the assay using primer LAMP SET 3 L3L1 consisting of SeqIDs 11 to
 16. 6. A LAMP assay as claimed in claim 1 for detection ofNeisseria meningitidis, the assay using LAMP primer SET 5 L5L1consisting of Seq IDs 24 to
 29. 7. A method for preparing an assay forthe detection of Neisseria meningitidis comprising utilizing a primer asset out in Table 1 as Sequence ID: 1 through Sequence ID: 69,individually or in combination with any of the other listed primers inthe preparation of the assay.
 8. Use of the primers as claimed in claim1 wherein the assay is a LAMP assay.
 9. A method for the detection ofNeisseria meningitidis serotypes A, B, C, Y or W 135 from a samplecomprising a LAMP assay for detection of Neisseria meningitidisserotypes A, B, C, Y or W 135, the test comprising at least one nucleicacid primer set capable of detecting Neisseria meningitidis in a LAMPbased molecular test, the primer set being chosen from the primer setslisted in Table 1 as LAMP SETS 1 to 12 comprising SEQUENCE IDs from ID:1 to ID:
 69. 10. A method of diagnosing meningococcal disease in apatient, the method comprising the steps of taking a sample from thepatient, accessing DNA in the sample, or extracting DNA from the sample,using at least one LAMP primer set as set out on Table 1 consisting ofprimer sequences chosen from sequence IDs 1 to 69 to amplify the DNA andtesting for amplification to confirm presence of Neisseria meningitidis.